Until about a decade ago, resolution in far-field light microscopy was thought to be limited to 200-280 nanometers in the focal plane, concealing details of sub-cellular structures and constraining its biological applications. Breaking this diffraction barrier by the seminal concept of stimulated emission depletion (“STED”) microscopy has made it possible to image biological systems at the nanoscale with light. STED microscopy and other members of reversible saturable optical fluorescence transitions (“RESOLFT”) family achieve a resolution greater than 10-fold beyond the diffraction barrier by engineering the microscope's point-spread function (“PSF”) through optically saturable transitions of the (fluorescent) probe molecules.
Current understanding of fundamental biological processes on the nanoscale (e.g., neural network formation, chromatin organization) is limited because these processes cannot be visualized, at a sub diffraction resolution, for an extended period, at imaging depths up to 50 microns. Current biological research at the sub-cellular level is constrained by the limits of spatial and temporal resolution in fluorescence microscopy. The diameter of most organelles is below the diffraction limit of light, limiting spatial resolution and concealing sub-structure. Although recent developments have improved spatial resolution and even overcome the traditional diffraction barriers, comparable improvements in temporal resolution are still needed.
Particle-tracking techniques can localize small objects (typically less than the diffraction limit) in live cells with sub-diffraction accuracy and track their movement over time. But conventional particle-tracking fluorescence microscopy cannot temporally resolve interactions of organelles, molecular machines, or even single proteins, which typically happen within milliseconds. The spatial localization accuracy of single particles in a fluorescence microscope is approximately proportional to spatial resolution divided by the total number of detected fluorescence photons from the particle in the absence of background and effects due to finite pixel size. For longer acquisition times more signal can be accumulated, hence increased temporal resolution requires a trade-off of decreased spatial localization accuracy.